1. Field of the Invention
The present invention relates to a human imunodeficiency virus (HIV) antigen. More particularly, the present invention is concerned with a substantially pure HIV antigen comprising a Gag-Env fusion protein consisting of a specific Gag peptide fused at its C-terminus to a specific Env peptide, which antigen not only exhibits excellent HIV antigenicity, but which can also be obtained at a level that has never been attained to date, and is also concerned with a method for producing the same. The HIV antigen of the present invention is useful as an active component for a diagnostic reagent, a vaccine, an antibody preparation and a therapeutic reagent for AIDS (acquired immune deficiency syndrome).
2. Discussion of Related Art
As is well know in the art, since the first AIDS patient was reported in 1981, the number of AIDS patients has been increasing in geometric progression. As of April 1992, the total number of AIDS patients is as large as about 500,000. Although research on the prevention and medical treatment of the disease have been extensively and intensively made throughout the world, no infallible preventive and therapeutic methods are in practical use. The global spread of AIDS without any infallible preventive and therapeutic methods is now a world-wide problem. On the other hand, the AIDS virus was first isolated and identified in 1983, and since then, research on AIDS in both the basic and clinical aspects has become active in the field of virology (see Nature, 326, 435-436, 1987). As a result, remarkable progress has been made in the diagnosis of AIDS, and immunodiagnostic reagents for use in the diagnosis and methods for producing the same are rapidly being improved. AIDS viruses have been isolated from humans, monkeys and cats. Of them, the virus isolated from humans is designated "human immunodeficiency virus (HIV)". HIV is broadly classified into HIV-1 and HIV-2. HIV-1 is spreading worldwide, i.e., in the U.S.A., Europe, Central Africa and other numerous countries of the world, while HIV-2 is mainly spreading only in West Africa. HIV is a spherical virus of from 100 to 140 nm in diameter which has an envelope (Env). The Env is comprised of transmembrane protein (gp41) and 70 to 80 peplomers (gp120) which form rod-shaped protrusions, each having a diameter of 15 nm and a height of about 9 nm, and which are present in the surface of the vital particle. In the core of the viral particle, two single strand RNA molecules of the viral genome form a complex with reverse transcriptase and structural proteins as the viral core, in which primer tRNA is present. The viral genome has a length of more than 9 kb and is comprised of about 10 different genes. Essentially, the viral genome is comprised of the following three major genes coding for the viral components essential for multiplication of the virus:
(1) gag (group-specific antigen) gene coding for p55 which is a precursor protein of three types of structural proteins p17, p24, and p15 of the viral core; PA1 (2) pol (polymerase) gene coding for a precursor of three different enzymes, i.e., protease, reverse transcriptase, and integrase; and PA1 (3) env (envelope) gene coding for gp160, which is a precursor of two types of glycoproteins, gp120 and gp41 forming the vital envelope.
These genes are arranged in the sequence of gag. pol . . . env in the direction from the 5'-end toward the 3'-end of the viral genome.
The remaining approximately seven other genes, so-called accessory genes, are believed to take part in the control of infection, multiplication, maturation of HIV, and the development of illness.
Various HIV antigens and enzymes essential for the basic studies of AIDS and for the development and production of therapeutic reagents, diagnostics and vaccines therefor can be produced by culturing HIV. However, the culturing of HIV is accompanied by the danger of fatal biohazard. Therefore, various studies and attempts have been made to develop a technique for the production of such antigens and enzymes in large quantity without culturing HIV. For example, with respect to both the gag and pol genes, various HIV antigens and enzymes, such as Gag proteins p17, p24, and p15 (see Japanese Patent Application Laid-Open Specification No. 4-117289) and pol gene products, e.g., protease, reverse transcriptase, and integrase (see Japanese Patent Application Laid-Open Specification No. 2-265481), have been successfully produced in high yield by a technique capable of expressing the genes in E. coli and processing the produced protein, and some of the antigens and enzymes have been put to practical use.
On the other hand, with respect to the expression of the env gene of HIV, the highly efficient expression of the env gene alone is extremely difficult, as compared to that of the gag and pol genes, although the reason has not yet been elucidated. Therefore, in many cases, the env gene of HIV is expressed in a chimeric form with a foreign gene, thereby producing the Env peptide as a protein in which the Env peptide is fused to a foreign peptide. For example, it is known to express the env gene in a chimeric form with a poliovirus gene, to thereby produce a fusion protein in which the Env peptide is fused to a poliovirus antigen peptide (see Journal of Virology, 65, 2875-2883, 1991). It is also known to express the env gene in a chimeric form with a gag gene by means of E. coli expression plasmid pEV-vrf, to thereby produce a Gag-Env fusion protein in which the Env peptide is fused to the Gag peptide (Analytical Biochemistry, 161, 370-379, 1987). In these cases, a peptide coded for by a foreign gene or a structural gene, which is positioned downstream of a promotor in an expression plasmid, is fused at its C-terminus to the Env peptide. When the Env peptide is fused to a foreign peptide, the Env peptide is likely to exhibit non-specificity in a reaction with test serum. Therefore, the Env peptide which is fused to a foreign peptide is inferior to a pure HIV antigen in quality and reliability for use as an HIV antigen. Furthermore, the Env peptide which is fused to a foreign peptide is not good in terms of production yield.
It is conceivable to cleave a fusion protein at the site of the junction of the Env peptide and a foreign peptide, in order to remove the foreign peptide. However, by this method, it is not possible to obtain the desired Env peptide in a foreign peptide-free, pure form in high yield and at low cost.
On the other hand, it is known to express the Env peptide as a Gag-Env fusion protein consisting of a Gag peptide fused to the Env peptide (see Japanese Patent Application Laid-Open Specification No. 1-179687; Virology, 180, 811-813, 1991 and European Patent Application Publication No. 307149). However, the yield of the conventional Gag-Env fusion protein is likely to be poor, thereby causing the production cost to be high. Furthermore, such a Gag-Env fusion protein is likely to be poor in antigenicity, so that its reliability as an HIV antigen is low.
Thus, the conventional HIV antigens are disadvantageous in that they are poor in quality, reliability and productivity. Therefore, a novel HIV antigen which is free from such problems has been much desired from a practical and commercial viewpoint, and the development of such a novel HIV antigen has been a task of great urgency in the art.
As mentioned above, the Env peptide has conventionally been produced in relatively large quantity as a fusion protein in which the Env peptide is fused to a foreign peptide, but the conventionally obtained Env peptide as a fusion protein is likely to exhibit non-specificity in an antigen-antibody reaction, so that the fusion protein is unsatisfactory in quality and reliability for use as an HIV antigen. Such a fusion protein is unsuitable for the practical diagnosis of AIDS. It should further be noted that the present invention has been attained by overcoming the serious problem of the prior art that even when the gag gene and env gene are fused to each other and expressed by conventional genetic engineering techniques, a Gag-Env fusion protein which is highly reliable as an HIV antigen can never be produced in high yield.